Strain improvement of <Emphasis Type="Italic">Trichoderma viride</Emphasis> for increased cellulase production by irradiation of electron and <Superscript>12</Superscript>C<Superscript>6+</Superscript>-ion beams

Objectives

To improve cellulase production and activity, Trichoderma viride GSICC 62010 was subjected to mutation involving irradiation with an electron beam and subsequently with a ¹²C⁶⁺-ion beam.

Results

Mutant CIT 626 was the most promising cellulase producer after preliminary and secondary screening. Soluble protein production and cellulase activities were increased mutifold. The optimum temperature, pH and culture time for the maximum cellulase production of the selected mutant were 35 °C, pH 5 and 6 days. The highest cellulase production was obtained using wheat bran. The prepared cellulases from T. viride CIT 626 had twice the hydrolytic performance with sawdust (83 %) than that from the parent strain (42.5 %). Furthermore, molecular studies demonstrated that there were some key mutation sites suggesting that some amino acid changes in the protein caused by base mutations had led to the enhanced cellulase production and activity.

Conclusions

Mutagenesis with electron and ¹²C⁶⁺-ion beams could be developed as an effective tool for improvement of cellulase producing strains.

     

Intercropping oilseed rape as a potential relay crop for enhancing the biological control of green peach aphids and aphid‐transmitted virus diseases

Tobacco viruses transmitted by green peach aphids, Myzus persicae (Sulzer) (Hemiptera: Aphididae), cause severe disease in flue‐cured tobacco, Nicotiana tabacum L. (Solanaceae), in China and throughout the world. Field experiments were conducted in 2016 and 2017 in Longyan City, Fujian Province, China, to determine whether M. persicae and aphid‐transmitted virus diseases are affected by intercropping of oilseed rape, Brassica napus L. (Brassicaceae), in tobacco fields. The results showed that, compared with those in monocultured fields, the densities of M. persicae and winged aphids in intercropped fields significantly decreased in both 2016 and 2017. In particular, the appearance of winged aphids was delayed by ca. 7 days. Moreover, the densities of Aphidius gifuensis Ashmead (Hymenoptera: Aphidiidae), a parasitoid of the aphid, significantly increased in 2016 and 2017. Accordingly, the incidence rates of aphid‐transmitted virus diseases (those caused by the cucumber mosaic virus, potato virus Y, and tobacco etch virus) significantly decreased in the intercropped fields in 2016 and 2017. Tobacco yields and monetary value significantly increased in 2016 (by 10–25 and 14–29%, respectively) and 2017 (by 17–22 and 22–34%, respectively). Consequently, our results suggest that intercropping oilseed rape in tobacco fields is a good approach to regulating and controlling aphids and tobacco mosaic viruses, for example potyvirus, and this intercropping can help control aphid‐transmitted virus diseases in tobacco.     

Stable overexpression of arginase I and ornithine transcarbamylase in HepG2 cells improves its ammonia detoxification

HepG2 is an immortalized human hepatoma cell line that has been used for research into bioartificial liver systems. However, a low level of ammonia detoxification is its biggest drawback. In this work, a recombinant HepG2 cell line with stable overexpression of human arginase I (hArgI) and human ornithine transcarbamylase (hOTC), HepG2/(hArgI + hOTC)4, was developed using a eukaryotic dual gene expression vector pBudCE4.1. (1) The hArgI and hOTC enzymatic activity in HepG2/(hArgI + hOTC)4 cells were higher than in the control cells. (2) The ammonia tolerance capacity of HepG2/(hArgI + hOTC)4 cells was three times that of HepG2 cells and 37.5% of that of primary human hepatocytes in cultivation. In the experiment of ammonia detoxification, HepG2/(hArgI + hOTC)4 cells produced 3.1 times more urea (at 180 mM NH₄Cl) and 3.1 times more glutamine (at 120 mM NH₄Cl and 15 mM glutamate) than HepG2 cells, reaching 63.1% and 36.0% that of primary human hepatocytes, respectively. (3) The hArgI and hOTC overexpression did not influence the growth of HepG2 cells and also promoted the expression of other ammonia detoxification associated proteins including glutamine synthetase (GS), arginase II (ArgII), arginosuccinate synthase (ASS) and arginosuccinate lyase (ASL) in HepG2 cells. This work illustrates that the modification reported here made significant progress in the improvement of HepG2 cell function and the HepG2/(hArgI + hOTC)4 cells will provide a better selection for the application of bioartificial liver system. J. Cell. Biochem. 113: 518–527, 2012. © 2011 Wiley Periodicals, Inc.     

犬细小病毒NS1 非结构蛋白可诱导细胞凋亡

【目的】研究犬细小病毒(Canine parvovirus,CPV)非结构蛋白NS1在CPV引起宿主细胞凋亡中的作用,初步探讨CPV引起细胞凋亡的机制。【方法】首先采用PCR方法从犬细小病毒基因组中扩增NS1编码基因,然后利用pcDNA3.1A质粒构建NS1真核表达载体pcDNA-NS1,并通过HEK293FT细胞瞬时表达NS1重组蛋白,用Western-blot检测以确定重组NS1蛋白能否在真核细胞中表达。然后用CPV感染和用pcDNA-NS1表达载体转染F81宿主细胞,通过AnnexinV/PI双染法检测磷脂酰丝氨酸外翻和通过化学发光法检测caspase-3/7活性,分析感染CPV或转染NS1基因对F81宿主细胞凋亡的影响。【结果】结果表明,本实验扩增的NS1基因序列与GenBank的序列一致,构建的表达载体结构正确,并能够介导NS1基因在真核细胞中表达。感染CPV和转染NS1基因均能诱导F81细胞膜磷脂酰丝氨酸外翻和明显提高细胞内caspase-3/7的活性,表明CPV和NS1蛋白均能引起细胞的凋亡。【结论】CPV诱导宿主细胞凋亡与其编码的NS1非结构蛋白有关。     

A synthetic wheat with 56 chromosomes derived fromTriticum turgidum andAegilops tauschii

By colchicine treatment of hybrids between Triticum turgidum and Aegilops tauschii (as seedlings), a fertile wheat plant (SHW-L2) carrying 56 chromosomes was artificially synthesized. At metaphase I of 50 pollen mother cells, the 56 chromosomes of the new wheat SHW-L2 showed a mean pairing configuration of 2.82 univalents, 6.18 rod bivalents, 19.39 ring bivalents, 0.5 trivalents, and 0.14 quadrivalents. Cytological analyses suggested that SHW-L2 had additional 7 pairs of chromosomes from the A and D genome besides the 42 chromosomes of common wheat. The special chromosome constitution of SHW-L2 may be derived from the chromosome doubling by the colchicine treatment of seedlings and then spontaneous doubling of gametes.     

Improving the accuracy of genomic prediction in Chinese Holstein cattle by using one-step blending

Background

The one-step blending approach has been suggested for genomic prediction in dairy cattle. The core of this approach is to incorporate pedigree and phenotypic information of non-genotyped animals. The objective of this study was to investigate the improvement of the accuracy of genomic prediction using the one-step blending method in Chinese Holstein cattle.

Findings

Three methods, GBLUP (genomic best linear unbiased prediction), original one-step blending with a genomic relationship matrix, and adjusted one-step blending with an adjusted genomic relationship matrix, were compared with respect to the accuracy of genomic prediction for five milk production traits in Chinese Holstein. For the two one-step blending methods, de-regressed proofs of 17 509 non-genotyped cows, including 424 dams and 17 085 half-sisters of the validation cows, were incorporated in the prediction model. The results showed that, averaged over the five milk production traits, the one-step blending increased the accuracy of genomic prediction by about 0.12 compared to GBLUP. No further improvement in accuracies was obtained from the adjusted one-step blending over the original one-step blending in our situation. Improvements in accuracies obtained with both one-step blending methods were almost completely contributed by the non-genotyped dams.

Conclusions

Compared with GBLUP, the one-step blending approach can significantly improve the accuracy of genomic prediction for milk production traits in Chinese Holstein cattle. Thus, the one-step blending is a promising approach for practical genomic selection in Chinese Holstein cattle, where the reference population mainly consists of cows.     

犬细小病毒VP2蛋白在真核细胞中的分泌表达及特性

摘要:【目的】利用真核细胞分泌表达犬细小病毒VP2蛋白和研究其特性。【方法】为构建犬细小病毒（Canine parvovirus, CPV）VP2基因的真核分泌型表达载体,首先通过酶切从含有人CD5信号肽序列的质粒中将CD5信号肽基因片段切出,将其连接到真核表达载体pcDNA3.1A的多克隆位点上,构建成pcDNA3.1-CD5sp质粒。然后再通过PCR方法从含有犬细小病毒VP2基因的质粒中扩增VP2基因,并将其插入到pcDNA3.1- CD5sp载体中CD5信号肽的下游,构建成VP2基因的真核分泌型表达载体pcDNA-CD5sp-VP2。经磷酸钙介导转染293T细胞,使其在真核细胞中进行分泌表达,并通过ELISA检测表达的VP2蛋白与犬转铁蛋白受体（TfR）结合的活性。【结果】序列分析结果表明,本实验构建的犬细小病毒VP2基因真核分泌型表达载体结构正确,将该表达载体转染的293T细胞,在培养基中通过Western-blot检测到有VP2重组蛋白的存在。经ELISA检测表明表达的重组VP2蛋白具有与犬转铁蛋白受体结合的活性。【结论】 利用人的CD5信号肽实现了犬细小病毒VP2蛋白在真核细胞中的分泌表达,表达的VP2蛋白具有与犬转铁蛋白受体结合的活性。     

A 55 K SNP array-based genetic map and its utilization in QTL mapping for productive tiller number in common wheat

Key message

A high-density genetic map constructed with a wheat 55 K SNP array was highly consistent with the physical map of this species and it facilitated the identification of a novel major QTL for productive tiller number.

Abstract

Productive tiller number (PTN) plays a key role in wheat grain yield. In this study, a recombinant inbred line population with 199 lines derived from a cross between ‘20828’ and ‘Chuannong16’ was used to construct a high-density genetic map using wheat 55 K single nucleotide polymorphism (SNP) array. The constructed genetic map contains 12,109 SNP markers spanning 3021.04 cM across the 21 wheat chromosomes. The orders of the genetic and physical positions of these markers are generally in agreement, and they also match well with those based on the 660 K SNP array from which the one used in this study was derived. The ratios of SNPs located in each of the wheat deletion bins were similar among the wheat 9 K, 55 K, 90 K, 660 K and 820 K SNP arrays. Based on the constructed maps, a novel major quantitative trait locus QPtn.sau-4B for PTN was detected across multi-environments in a 0.55 cM interval on 4B and it explained 17.23–45.46% of the phenotypic variance. Twenty common genes in the physical interval between the flanking markers were identified on chromosome 4B of ‘Chinese Spring’ and wild emmer. These results indicate that wheat 55 K SNP array could be an ideal tool in primary mapping of target genes and the identification of QPtn.sau-4B laid a foundation for the following fine mapping and cloning work.